Sydney E. Everhart

Influence of bark pH on the occurrence and distribution of tree canopy myxomycete species

This study compares the occurrence and distribution of myxomycete species in the canopy of living trees and neighboring grapevines. Corticolous myxomycetes of three temperate forests in southeastern USA were studied on six tree species (30 trees) and grapevines (30 vines) to determine distribution and occurrence of myxomycete species relating to geographic location, host species, and bark pH. The double-rope climbing technique was used to access the canopy and sample bark up to 16.5 m. Bark samples were examined in 580 moist chamber cultures and 44 myxomycete species were identified representing 21 genera, averaging 3.0 ± 2.1 species per sample site. Jaccard’s coefficient determined community similarity between five individuals of six tree species, Acer saccharum, Fraxinus americana, Liquidambar styraciflua, Liriodendron tulipifera, Platanus occidentalis and Tsuga canadensis, and neighboring grapevines, Vitis aestivalis and V. vulpina. Vertical variation in species richness was significantly different only for Platanus occidentalis and might be attributable to flaking of bark with increasing height in the canopy. Tsuga canadensis and neighboring grapevines had greatest community similarity. Cribraria violacea was observed on all tree and grapevine species except T. canadensis and neighboring grapevines. Occurrence and species assemblages of myxomycetes were associated with bark pH, not geographic location. Bark of V. aestivalis (pH 4.5) was more acidic than neighboring T. canadensis (pH 4.1), compared to grapevines of the same species neighboring other tree species. Results indicated that most species are not regionally restricted, and although some myxomycetes are associated with a certain pH range, others develop on any substratum. Future research protocols for corticolous myxomycetes should emphasize sampling adequate amounts of substrata in a local region from different host species that have a wide range of bark pH, ensuring a representative sample of species for an entire region.

Characterization of Three-Dimensional Spatial Aggregation and Association Patterns of Brown Rot Symptoms within Intensively Mapped Sour Cherry Trees

BACKGROUND AND AIMS Characterization of spatial patterns of plant disease can provide insights into important epidemiological processes such as sources of inoculum, mechanisms of dissemination, and reproductive strategies of the pathogen population. Whilst two-dimensional patterns of disease (among plants within fields) have been studied extensively, there is limited information on three-dimensional patterns within individual plant canopies. Reported here are the detailed mapping of different symptom types of brown rot (caused by Monilinia laxa) in individual sour cherry tree (Prunus cerasus) canopies, and the application of spatial statistics to the resulting data points to determine patterns of symptom aggregation and association. METHODS A magnetic digitizer was utilized to create detailed three-dimensional maps of three symptom types (blossom blight, shoot blight and twig canker) in eight sour cherry tree canopies during the green fruit stage of development. The resulting point patterns were analysed for aggregation (within a given symptom type) and pairwise association (between symptom types) using a three-dimensional extension of nearest-neighbour analysis. KEY RESULTS Symptoms of M. laxa infection were generally aggregated within the canopy volume, but there was no consistent pattern for one symptom type to be more or less aggregated than the other. Analysis of spatial association among symptom types indicated that previous years twig cankers may play an important role in influencing the spatial pattern of current years symptoms. This observation provides quantitative support for the epidemiological role of twig cankers as sources of primary inoculum within the tree. CONCLUSIONS Presented here is a new approach to quantify spatial patterns of plant disease in complex fruit tree canopies using point pattern analysis. This work provides a framework for quantitative analysis of three-dimensional spatial patterns within the finite tree canopy, applicable to many fields of research.

Spatio-temporal Patterns of Pre-harvest Brown Rot Epidemics within Individual Peach Tree Canopies

Tree canopies are architecturally complex and pose several challenges for measuring and characterizing spatial patterns of disease. Recently developed methods for fine-scale canopy mapping and three-dimensional spatial pattern analysis were applied in a 3-year study to characterize spatio-temporal development of pre-harvest brown rot of peach, caused by Monilinia fructicola, in 13 trees of different maturity classes. We observed a negative correlation between an index of disease aggregation and disease incidence in the same tree (r = −0.653, P < 0.0001), showing that trees with higher brown rot incidence had lower aggregation of affected fruit in their canopies. Significant (P ≤ 0.05) within-canopy aggregation among symptomatic fruit was most pronounced for early-maturing cultivars and/or early in the epidemic. This is consistent with the notion of a greater importance of localized, within-tree sources of inoculum at the beginning of the epidemic. Four of five trees having >10 blossom blight symptoms per tree showed a significant positive spatial association of pre-harvest fruit rot to blossom blight within the same canopy. Spatial association analyses further revealed one of two outcomes for the association of new fruit rot symptoms with previous fruit rot symptoms in the same tree, whereby the relationship was either not significant or exhibited a significant negative association. In the latter scenario, the newly diseased fruit were farther apart from previously symptomatic fruit than expected by random chance. This unexpected result could have been due to uneven fruit ripening in different sectors of the canopy, which could have affected the timing of symptom development and thus led to negative spatial associations among symptoms developing over time in a tree.

Fine-Scale Genetic Structure of Monilinia fructicola During Brown Rot Epidemics Within Individual Peach Tree Canopies

The purpose of this study was to determine the fine-scale genetic structure of populations of the brown rot pathogen Monilinia fructicola within individual peach tree canopies to better understand within-tree plant pathogen diversity and to complement previous work on spatiotemporal development of brown rot disease at the canopy level. Across 3 years in a total of six trees, we monitored disease development, collected isolates from every M. fructicola symptom during the course of the season, and created high-resolution three-dimensional maps of all symptom and isolate locations within individual canopies using an electromagnetic digitizer. Each canopy population (65 to 173 isolates per tree) was characterized using a set of 13 microsatellite markers and analyzed for evidence of spatial genetic autocorrelation among isolates during the epidemic phase of the disease. Results showed high genetic diversity (average uh=0.529) and high genotypic diversity (average D=0.928) within canopies. The percentage of unique multilocus genotypes within trees was greater for blossom blight isolates (78.2%) than for fruit rot isolates (51.3%), indicating a greater contribution of clonal reproduction during the preharvest epidemic. For fruit rot isolates, between 54.2 and 81.7% of isolates were contained in one to four dominant clonal genotypes per tree having at least 10 members. All six fruit rot populations showed positive and significant spatial genetic autocorrelation for distance classes between 0.37 and 1.48 m. Despite high levels of within-tree pathogen diversity, the contribution of locally available inoculum combined with short-distance dispersal is likely the main factor generating clonal population foci and associated spatial genetic clustering within trees.

Fungicide-induced transposon movement in Monilinia fructicola

Repeated applications of fungicides with a single mode of action are believed to select for pre-existing resistant strains in a pathogen population, while the impact of sub-lethal doses of such fungicides on sensitive members of the population is unknown. In this study, in vitro evidence is presented that continuous exposure of Monilinia fructicola mycelium to some fungicides can induce genetic change in form of transposon transposition. Three fungicide-sensitive M. fructicola isolates were exposed in 12 weekly transfers of mycelia to a dose gradient of demethylation inhibitor fungicide (DMI) SYP-Z048 and quinone outside inhibitor fungicide (QoI) azoxystrobin in solo or mixture treatments. Evidence of mutagenesis was assessed by monitoring Mftc1, a multicopy transposable element of M. fructicola, by PCR and Southern blot analysis. Movement of Mftc1 was observed following azoxystrobin and azoxystrobin plus SYP-Z048 treatments in two of the three isolates, but not in the non-fungicide-treated controls. Interestingly, the upstream promoter region of MfCYP51 was a prime target for Mftc1 transposition in these isolates. Transposition of Mftc1 was verified by Southern blot in two of three isolates from another, similar experiment following prolonged, sublethal azoxystrobin exposure, although in these isolates movement of Mftc1 in the upstream MfCYP51 promoter region was not observed. More research is warranted to determine whether fungicide-induced mutagenesis may also happen under field conditions.

Population structure and phenotypic variation of Sclerotinia sclerotiorum from dry bean (Phaseolus vulgaris) in the United States

The ascomycete pathogen Sclerotinia sclerotiorum is a necrotrophic pathogen on over 400 known host plants, and is the causal agent of white mold on dry bean. Currently, there are no known cultivars of dry bean with complete resistance to white mold. For more than 20 years, bean breeders have been using white mold screening nurseries (wmn) with natural populations of S. sclerotiorum to screen new cultivars for resistance. It is thus important to know if the genetic diversity in populations of S. sclerotiorum within these nurseries (a) reflect the genetic diversity of the populations in the surrounding region and (b) are stable over time. Furthermore, previous studies have investigated the correlation between mycelial compatibility groups (MCG) and multilocus haplotypes (MLH), but none have formally tested these patterns. We genotyped 366 isolates of S. sclerotiorum from producer fields and wmn surveyed over 10 years in 2003–2012 representing 11 states in the United States of America, Australia, France, and Mexico at 11 microsatellite loci resulting in 165 MLHs. Populations were loosely structured over space and time based on analysis of molecular variance and discriminant analysis of principal components, but not by cultivar, aggressiveness, or field source. Of all the regions tested, only Mexico (n = 18) shared no MLHs with any other region. Using a bipartite network-based approach, we found no evidence that the MCGs accurately represent MLHs. Our study suggests that breeders should continue to test dry bean lines in several wmn across the United States to account for both the phenotypic and genotypic variation that exists across regions.

Effects of Sublethal Fungicides on Mutation Rates and Genomic Variation in Fungal Plant Pathogen, Sclerotinia sclerotiorum

Pathogen exposure to sublethal doses of fungicides may result in mutations that may represent an important and largely overlooked mechanism of introducing new genetic variation into strictly clonal populations, including acquisition of fungicide resistance. We tested this hypothesis using the clonal plant pathogen, Sclerotinia sclerotiorum. Nine susceptible isolates were exposed independently to five commercial fungicides with different modes of action: boscalid (respiration inhibitor), iprodione (unclear mode of action), thiophanate methyl (inhibition of microtubulin synthesis) and azoxystrobin and pyraclostrobin (quinone outside inhibitors). Mycelium of each isolate was inoculated onto a fungicide gradient and sub-cultured from the 50–100% inhibition zone for 12 generations and experiment repeated. Mutational changes were assessed for all isolates at six neutral microsatellite (SSR) loci and for a subset of isolates using amplified fragment length polymorphisms (AFLPs). SSR analysis showed 12 of 85 fungicide-exposed isolates had a total of 127 stepwise mutations with 42 insertions and 85 deletions. Most stepwise deletions were in iprodione- and azoxystrobin-exposed isolates (n = 40/85 each). Estimated mutation rates were 1.7 to 60-fold higher for mutated loci compared to that expected under neutral conditions. AFLP genotyping of 33 isolates (16 non-exposed control and 17 fungicide exposed) generated 602 polymorphic alleles. Cluster analysis with principal coordinate analysis (PCoA) and discriminant analysis of principal components (DAPC) identified fungicide-exposed isolates as a distinct group from non-exposed control isolates (PhiPT = 0.15, P = 0.001). Dendrograms based on neighbor-joining also supported allelic variation associated with fungicide-exposure. Fungicide sensitivity of isolates measured throughout both experiments did not show consistent trends. For example, eight isolates exposed to boscalid had higher EC50 values at the end of the experiment, and when repeated, only one isolate had higher EC50 while most isolates showed no difference. Results of this support the hypothesis that sublethal fungicide stress increases mutation rates in a largely clonal plant pathogen under in vitro conditions. Collectively, this work will aid our understanding how non-lethal fungicide exposure may affect genomic variation, which may be an important mechanism of novel trait emergence, adaptation, and evolution for clonal organisms.

Effect of four training systems on the temporal dynamics of downy mildew in two grapevine cultivars in southern Brazil

The aim of this study was to evaluate the temporal dynamics of downy mildew epidemics under four training systems: vertical shoot positioning (VSP), Geneva Double Curtain (GDC), Simple Curtain (SC) and Tendone in two cultivars (‘Cabernet Sauvignon’ and ‘Merlot’). Experiments were conducted at commercial vineyards in southern Brazil, during the 2012–2013 and 2013–2014 growing seasons. Downy mildew incidence and severity were quantified on a weekly basis from the first symptoms appearance until harvest. Training systems and cultivars were compared based on the following variables of downy mildew progress curves: a) beginning of symptoms appearance (BSA); b) maximum disease incidence and severity (Imax, Smax); c) time to reach maximum disease incidence/severity (TRMDI and TRMDS); and d) area under incidence and severity disease progress curve (AUIDPC and AUSDPC). The training systems significantly affected AUSDPC and Smax. AUSDPC was lowest in the VSP system in all cultivars and growing seasons. In addition, ‘Merlot’ showed significantly lower AUIDPC and AUSDPC than ‘Cabernet Sauvignon’ in both growing seasons. The Tendone and GDC systems showed significantly higher AUSDPC in all cultivars and growing seasons. Our results suggested that the use of the VSP system contributes to reduce downy mildew severity on ‘Merlot’ and ‘Cabernet Sauvignon’ in southern Brazil.

Effect of Fungicide Applications on Monilinia fructicola Population Diversity and Transposon Movement

In this study, we investigated whether fungicide-induced mutagenesis previously reported in Monilinia fructicola could accelerate genetic changes in field populations. Azoxystrobin and propiconazole were applied to nectarine trees at weekly intervals for approximately 3 months between bloom and harvest in both 2013 and 2014. Fungicides were applied at half-label rate to allow recovery of isolates and to increase chances of sublethal dose exposure. One block was left unsprayed as a control. In total, 608 single-spore isolates were obtained from blighted blossoms, cankers, and fruit to investigate phenotypic (fungicide resistance) and genotypic (simple-sequence repeat [SSR] loci and gene region) changes. In both years, populations from fungicide-treated and untreated fruit were not statistically different in haploid gene diversity (P = 0.775 for 2013 and P = 0.938 for 2014), allele number (P = 0.876 for 2013 and P = 0.406 for 2014), and effective allele number (P = 0.861 for 2013 and P = 0.814 for 2014). Isolates from blossoms and corresponding cankers of fungicide treatments revealed no changes in SSR analysis or evidence for induced Mftc1 transposon translocation. No indirect evidence for increased genetic diversity in the form of emergence of reduced sensitivity to azoxystrobin, propiconazole, iprodione, and cyprodinil was detected. High levels of population diversity in all treatments provided evidence for sexual recombination of this pathogen in the field, despite apparent absence of apothecia in the orchard. Our results indicate that fungicide-induced, genetic changes may not occur or not occur as readily in field populations as they do under continuous exposure to sublethal doses in vitro.

Upper Canopy Collection and Identification of Grapevines (Vitis) from Selected Forests in the Southeastern United States

Abstract Woody grapevines (Vitis spp.) are common in the deciduous forests of the southeastern United States. Their growth habit makes leaf collection challenging and polymorphic leaves make identification of species difficult. Mature grapevines can grow up to 48 cm in diameter at breast height and reach the upper canopy of trees more than 35 m in height. Leaf morphology is the most readily available character used for species identification. However, most mature grapevines do not produce leaves below the upper canopy and if they do, these leaves are morphologically indistinguishable from other species. In order to sample leaves from mature grapevines, the doubled rope climbing method was used to access the canopy in Great Smoky Mountains National Park in Tennessee, Daniel Boone National Forest and Berea College Forest in Kentucky, and Ha Ha Tonka State Park in Missouri. Leaf voucher specimens were collected from the upper canopy and used to create a modified key to species for those regions. The purposes of this paper are to report a new method for collecting grapevine leaf vouchers from the upper canopy of trees, to present a modified key used for identifying dried leaf vouchers of Vitis species, and to present a discussion of the possible utility of this research for future studies.